DESIGNING A SYNTHETIC ORGANISM Asfa A S (HT080934L) Vasanth Natarajan (HT081073M) Department of Chemical & Biomolecular Engineering National University of Singapore
Jan 01, 2016
DESIGNING A SYNTHETIC ORGANISM
Asfa A S (HT080934L)
Vasanth Natarajan (HT081073M)
Department of Chemical & Biomolecular EngineeringNational University of Singapore
SYNTHETIC BIOLOGY
Origin of Life
Designer Cells
Minimal Genome
Recreate Life
Vision of “SYNTHETIC BIOLOGY “
Synthetic Biology
Synthetic biology is an ambitious and relatively new field of biology that hopes to recreate life. The first and foremost challenge in creating 'life in lab' lies in identifying the minimum essential components that can take on the essential properties of a living organism
What Defines LIFE
LIVING CELL
Autonomous Replication
Darwinian Evolution
Continued growth and division dependent on input of small molecules and energy
Genetic and phenotypic variation for survival and reproduction
Bottom Up Approach Top Down Approach
Design a protocell
Semi-synthetic
Current Strategies
Synthesizing cell from scratch
Strip down the genes of an existing cell to bare minimum enough to sustain life
Synthesizing Life – Bottom Up Approach
Assembly of single lipid molecules/micelles
Gradual growth
Environmental factors to control division
RNA - store information
RNA – RNA polymerase – replicate its own sequence
2 RNA molecules – simplest cell
Szostak et al. , Nature 2001
Minimal Genome Concept
Aims to strip down a present day bacterium to its minimum essential components pertaining to replication, transcription and translation machinery.
Understand the basic components of the cell that makes it living.
Provides a template genome that can be used to recreate life
A less complex cell that can be reliably modeled and engineered to meet our requirements.
Essential Genes – A Comparative Study
0
50
100
150
200
250
300
350
400
450
Studies on Minimal Genome
Ess
enti
al G
enes
Craig Venter, 2005
Ehrlich SD,2003
Gil,2004
Koonin,1996
Mycoplasma genitalium – 482 protein coding genes – smallest genome
Craig Venter,2005 – 382 protein coding genes + 5 paralogous families – Transposon mutagenesis
Ehrilch SD, 2003 – 271 essential genes in Bacillus subtilis – Gene knock out by non replicating plasmid
Gil, 2004 – 206 essential genes – Comparison of Endosymbioints - Predicted
Koonin, 1996 – 256 essential genes – Comparison of M.genitalium and H.influenzae - Predicted
Functional Groups
Main Roles Craig Venter, 2005
Ehrlich SD, 2003
Koonin, 1996
Gil, 2004
Intermediary metabolism 4 2 0 2 Transport and binding proteins
35 35 7 32
Protein fate 24 16 3 10 Transcription 12 7 1 3 Cell envelope 35 35 23 35 Hypothetical proteins 47 47 43 45 Unknown function 37 36 20 29 Nucleosides and nucleotides 16 8 1 8 Energy metabolism 29 21 1 10 Protein synthesis 95 11 7 10 cell/organism defense 1 1 1 1 Synthesis of cofactors and carriers
7 2 1 3
Cellular processes 6 5 0 2 Fats and phospholipid metabolism
6 3 2 5
Regulatory function 2 2 1 2 DNA metabolism 25 12 3 11
Essential Genes – A Conclusive List
Different studies come up with a different number of essential genes.
Computation - Underestimates minimal genes - accounts only those genes that have been conserved in evolution.
Transposon mutagenesis - Over estimates the genes – Classifies genes that slow down growth as essential and essential genes that tolerate mutation as non essential.
Antisense RNA - limited success rates
Most mutants produced are single mutants – synthetic lethality may not be accounted
Construction of a single cell with systematic combination of all the mutations in a single
strain is beyond the scope of present day technology.
Designing a Synthetic Organism
Determine the minimal genes
Synthesize and assemble the
genome
Genome Transplantation
SYNTHETIC ORGANISM
Transposon Mutagenesis
Gene knockout using non replicating plasmid insertions
Antisense RNA
Computationally Predicted
Into a suitable propagating cell that can take up the genome
STRATEGY
Engineer the genome/add new
functions
Success so far…
Infectious Virus Completely Synthesized – World’s First Artificial Organism - 2002
3026 bp 2682 bp1895 bp
cDNA - T7 RNA polymerase promoter constructed from 3 overlapping DNA fragments.
Each fragment - overlapping 400-600 bp.
Each segment – 69 nt of + and – sequences
cDNA transcribed – Infectious RNA
Infection demonstrated in mice.
SYNTHETIC RNA => TRANSLATED => REPLICATED =>ENCAPSIDATED INTO NEW COAT PROTEINS
Cello et al. Science, 2002
In the Future…
Mycoplasma laboratorium
Synthetic Genome
Only essential 382 genes
Complete synthesis, cloning and sequential assembly
Immortal synthetic organism
Military Purpose – Pentagon
Self killing switch
Synthetic Algae
Biofuel
Synthetic Genomics
Biofuel – A dream in the making
Goals
Seeks alternatives to fossil fuels Sustainability Cost reduction
Challenges
Microorganisms can be designed to make useful materials from renewable materials (Sustainability) - to seek alternatives to fossil fuels.
In this case, designing a set of chemical pathways which allows conversion of natural or waste materials for the production of Biofuels .
Biofuel – A dream in the making (contd.)
ZM - Z.mobilisSC - S.cerevisiaeEC - E.coli
adh,pdc,pfl
Genes that are important for ethanol production
How to design a synthetic organism by adding new functions to the existing genome ?
Biofuel – A Strategy for Designing synthetic organism
Identified Essential gene list
Add new genes to the
existing prototype and
assemble genome
Screen for viability of
cell , maximum replication and higher
ethanol production
Adh,pdc,pfl
Synthetic organism
which produces
ethanol with minimal genes
Genome Transplanta
tion
success
NO YESWhat next ?
Add few more imp
genes
Biomedical Applications
Devices- For example, for tissue regeneration or tissue repair complex molecular devices can be developed.
- Another example could be development of macromolecular assemblies to sense the damage in blood vessels and repair them.
Novel Drug Release Technology
Smart Drugs -----> Synthetic molecular ensemble
Encapsulates drug in an inactive form.
Sensing disease indicators
The programmed module will make a decision
Activates the drug . (Active only in cells affected by disease)
Inactive form
Inactive form
Active form
Programmed ModuleDisease
Indicators
Environmental Applications
Bioremediation:
Treatment of environmental contaminants via biological systems.
Rational modification of bacteria and other microorganisms to eliminate toxic waste from soil.
For certain chemicals for which clean up is difficult, novel organisms with specific wiring can be used.
Biosensing : Detect biotoxins Helps in detecting toxin levels in environment
The Hindering Factor
How to overcome ? Bio engineered systems
remains noisy
Not easier to predict accurately how a new system will behave
Engineered organisms capable of self replication and evolution
Expensive , Unreliable and adhoc biological systems
Obstacles
FORSEEN RISKS
Some of the risks are indefinable at present – we cannot anticipate certain risks at this early stage
Accidental release of harmful organism - Extinction of existing species - Endemic - Damaging/Disrupting the habitat ( Upset natural balance)
Purposeful Design and release of harmful organism – Bioterrorism
Bio-hacker culture
Control Measures
To educate and train a responsible generation of bioengineers and scientists
Working with approved research facilitiesControls and regulations can be imposed on part
suppliers (eg . screening of oligonucleotides )Strict laws and policies to be imposed. Incorporating novel genetic codes for high risk
organisms to avoid tampering.
Conclusion
Synthetic Biology – Greatest existing challenge
Synthetic and semi-synthetic approaches.
Discerning the minimal genome enhances better understanding of cells
Engineered organism can be used for various applications in fields of biomedicine and environment
Potential risks and hazards not clear
Key References
Cello J et al. Chemical Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the Absence of Natural Template. Science(2002), 297
Smith et al. Complete Chemical Synthesis, Assembly and Cloning of a Mycoplasma genitalium Genome. Science(2008), 319
Koonin et al. A Minimal Gene Set for Cellular Life Derived by Comparison of Complete Bacterial Genomes. PNAS(1996),93
Venter C.J et al. Essential Genes of a Minimal Bacterium. PNAS(2006),103
Szostak et al. Synthesizing Life. Nature(2001),409
Ehrlich SD et al. Essential Bacillus subtilis genes.PNAS(2003),100
Gil et al. Determination of the Core of a Minimal Bacterial Gene Set. Microbiology and Molecular Biology Reviews(2004),68